Changes in intracellular cations during the cell cycle in HeLa cells

Intracellular Na+, K+, and Mg2+ concentrations have been measured during the HeLa cell cycle and compared with changes in oxygen utilization and macromolecular synthesis. Cell water content remains relatively constant at 79 +/- 1% during the cell cycle. A biphasic change in intracellular Na+ occurs with low values as cells reach peak S phase and again in early G1. The decrease in S coincides with an increase in cell volume during increased macromolecular synthesis. The fall in intracellular Na+ during mitosis/early G1 coincides with decreased energy utilization as macromolecular synthesis decreases with a continued decrease in [Na+]i in G1 corresponding to a period of increasing cell volume and an increase in protein synthesis. Intracellular Na+ is relatively high during late S/G2 when phosphate incorporation into protein and phospholipid is maximal. Intracellular K+ concentrations largely parallel intracellular Na+ levels although the intracellular K+:Na+ ratio is significantly lower as the cell volume increases during late G2/mitosis. Additions of a Na+-pump inhibitor (strophanthidin) not only caused a rise in [Na+]i and fall in [K+]i but also inhibited protein synthesis. Conversely, addition of a protein synthesis inhibitor (cycloheximide) blocked amino acid incorporation and produces a fall in intracellular Na+ levels. These findings indicate that intracellular Na+ and K+ play an important role in regulating cell hydration during the cell cycle and that changes in Na+, K+-ATPase activity, synthesis and/or utilization of high energy phosphate compounds, fluid phase turnover (endocytosis), Na+:H+ exchange (pHi), Donnan forces, and ionic adsorption may all be involved.     

Mechanism for pre-B cell loss in VH-mutant rabbits

Pre-BCR signaling is a critical checkpoint in B cell development in which B-lineage cells expressing functional IgH μ-chain are selectively expanded. B cell development is delayed in mutant ali/ali rabbits because the a-allotype encoding V(H)1 gene, which is normally used in VDJ gene rearrangements in wt rabbits, is deleted, and instead, most B-lineage cells use the a-allotype encoding V(H)4 gene [V(H)4(a)], which results in a severe developmental block at the pre-B cell stage. We found that V(H)4(a)-utilizing pre-B cells exhibit reduced pre-BCR signaling and do not undergo normal expansion in vitro. Transduction of murine 38B9 pre-B cells with chimeric rabbit-VDJ mouse-Cμ encoding retroviruses showed V(H)4(a)-encoded μ-chains do not readily form signal-competent pre-BCR, thereby explaining the reduction in pre-BCR signaling and pre-B cell expansion. Development of V(H)4(a)-utilizing B cells can be rescued in vivo by the expression of an Igκ transgene, indicating that V(H)4(a)-μ chains are not defective for conventional BCR formation and signaling. The ali/ali rabbit model system is unique because V(H)4(a)-μ chains have the capacity to pair with a variety of conventional IgL chains and yet lack the capacity to form a signal-competent pre-BCR. This system could allow for identification of critical structural parameters that govern pre-BCR formation/signaling.     

Purification and characterization of monodehydroascorbate reductase from soybean root nodules.

Soybean (Glycine max (L.) Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle as an important defense against activated forms of oxygen. A key enzyme in this cycle--monodehydroascorbate reductase (MR)--was purified 646-fold and appeared as a single band on SDS-PAGE with silver or Coomassie blue staining. Purified MR contained 0.7 mol FAD/mol enzyme and had a specific activity of 288 mumol NADH oxidized.min-1.mg protein-1. The enzyme was a single subunit occurring as two isozymes (MR I and MR II) with Mr values of 39,000 and 40,000. Isoelectric focusing revealed that each isozyme consisted of two forms with pl values of 4.6 to 4.7. Ferricyanide and 2,6-dichlorophenol-indophenol were effective as electron acceptors. The purified enzyme did not possess leghemoglobin reductase activity. Inhibition by p-chloromercuribenzoate indicated the involvement of a thiol group in MR activity. The Km values were 5.6, 150, and 7 microM for NADH, NADPH, and monodehydroascorbate, respectively. The pH optimum was 8 to 9. The N-terminal sequence of 10 amino acids of MR II had little homology to known protein sequences.     

Group size and stability: Why do gibbons and spider monkeys differ?

Gibbons and spider monkeys have similar diets, body size, and locomotor patterns. They are therefore expected to be subject to similar socioecological rules. However their grouping patterns differ. Gibbons live in small stable groups, whereas spider monkey form unstable sub-groups that vary from small to large during different seasons. If similar principles apply to the two species, food abundance should vary more for spider monkeys than for gibbons; food density should be similar for the two species when spider monkey sub-groups are the same size as gibbon groups; and the highest level of food abundance should be higher for spider monkeys than for gibbons. These predictions are upheld for a comparison of particular populations ofHylobates muelleri andAteles geoffroyi.     

88 Comparison of monovalent and divalent ion distributions around a DNA duplex with molecular dynamic simulation and Poisson–Boltzmann approach

Ion interactions with nucleic acids (both DNA and RNA) are an important and evolving field of investigation. Positively charged cations may interact with highly negatively charged nucleic acids via simple electrostatic interactions to help screen the electrostatic repulsion along the nucleic acids and assist their folding and/or compaction. Cations may also bind at specific sites and become integral parts of the structures, possibly playing important enzymatic roles. Two popular methods for computationally exploring a nucleic acid’s ion atmosphere are atomistic molecular dynamics (MD) simulations and the Poisson–Boltzmann (PB) equation. In general, monovalent ion results obtained from MD simulations and the PB equation agree well with experiment. However, Bai et al. (2007) observed discrepancies between experiment and the PB equation while examining the competitive binding of monovalent and divalent ions, with more significant discrepancies for divalent ions. The goal of this project was to thoroughly investigate monovalent (Na⁺) and divalent (Mg²⁺) ion distributions formed around a DNA duplex with MD simulations and the PB equation. We simulated three different cation concentrations, and matched the equilibrated bulk ion concentration for our theoretical calculations with the PB equation. Based on previous work, our Mg²⁺ ions were fully solvated, the expected state of Mg²⁺ ions when interacting with a duplex, when the production simulations began and remained throughout the simulations (Kirmizialtin, 2010; Robbins, 2012). Na⁺ ion distributions and number of Na⁺ ions within 10?Å of the DNA obtained from our two methods agreed well. However, results differed for Mg²⁺ ions, with a lower number of ions within the cut-off distance obtained from the PB equation when compared to MD simulations. The Mg²⁺ ion distributions around the DNA obtained via the two methods also differed. Based on our results, we conclude that the PB equation will systematically underestimate Mg²⁺ ions bound to DNA, and much of this deviation is attributed to dielectric saturation associated with high valency ions.     

Pre-induction priming of the uterine cervix with oral prostaglandin E2 and a placebo

A double blind study was undertaken to determine the effectiveness or oral prostaglandin E2 as a means of improving the pelvic score prior to induction of labour. 48 patients who were greater than 37 were gestation and who had Bishop scores of less than 6 entered the study. Ten tablets were given on an hourly regime. Of 25 patients in the prostaglandin group, 17 were considered successes (68.0%), whereas of 23 patients who received a placebo, 9 were successes (39.1%). No adverse effects were recorded. Prostaglandin E2 is therefore considered a safe and effective method for priming the unfavourable cervix prior to induction of labour.     

Association of p60fyn with middle tumor antigen in murine polyomavirus-transformed rat cells.

Rabbit antisera raised against human FYN-specific peptides were used to evaluate the expression of the fyn gene product in normal and murine polyomavirus middle tumor antigen (MTAg)-transformed rat cells. The antisera were capable of detecting p60fyn in both normal and MTAg-transformed cells. Two different antisera directed against unique p60fyn sequences were found to detect p60fyn-MTAg complexes in cell lysates from the MTAg-transformed cells. The MTAg molecules immunoprecipitated by FYN antisera were phosphorylated on tyrosine during immune-complex kinase reactions at sites similar to those found on MTAg in complexes with pp60c-src. Whereas the abundance of p60fyn was estimated to be less in the MTAg-transformed cells than in their normal counterparts, the specific activities of p60fyn molecules in the normal and transformed cells were similar.     

Male secondary sexual structures and the systematics of the Thereus oppia species group (Lepidoptera,Lycaenidae, Eumaeini)

The Thereus oppia species group includes species with and without a scent pad, which is a histologically and morphologically characterized male secondary sexual structure on the dorsal surface of the forewing. To assess the hypothesis that these structures are lost evolutionarily, but not regained (Dollo’s Law), the taxonomy of this species group is revised. Thereus lomalarga sp. n., and Thereus brocki sp. n., are described. Diagnostic traits, especially male secondary structures, within the Thereus oppia species group are illustrated. Distributional and biological information is summarized for each species. Three species have been reared, and the caterpillars eat Loranthaceae. An inferred phylogeny is consistent with the hypothesis that scent pads in the Thereus oppia species group have been lost evolutionarily twice (in allopatry), and not re-gained.     

Predicting demographic group structures based on DNA sequence data

The ability to infer relationships between groups of sequences, either by searching for their evolutionary history or by comparing their sequence similarity, can be a crucial step in hypothesis testing. Interpreting relationships of human immunodeficiency virus type 1 (HIV-1) sequences can be challenging because of their rapidly evolving genomes, but it may also lead to a better understanding of the underlying biology. Several studies have focused on the evolution of HIV-1, but there is little information to link sequence similarities and evolutionary histories of HIV-1 to the epidemiological information of the infected individual. Our goal was to correlate patterns of HIV-1 genetic diversity with epidemiological information, including risk and demographic factors. These correlations were then used to predict epidemiological information through analyzing short stretches of HIV-1 sequence. Using standard phylogenetic and phenetic techniques on 100 HIV-1 subtype B sequences, we were able to show some correlation between the viral sequences and the geographic area of infection and the risk of men who engage in sex with men. To help identify more subtle relationships between the viral sequences, the method of multidimensional scaling (MDS) was performed. That method identified statistically significant correlations between the viral sequences and the risk factors of men who engage in sex with men and individuals who engage in sex with injection drug users or use injection drugs themselves. Using tree construction, MDS, and newly developed likelihood assignment methods on the original 100 samples we sequenced, and also on a set of blinded samples, we were able to predict demographic/risk group membership at a rate statistically better than by chance alone. Such methods may make it possible to identify viral variants belonging to specific demographic groups by examining only a small portion of the HIV-1 genome. Such predictions of demographic epidemiology based on sequence information may become valuable in assigning different treatment regimens to infected individuals.     

Kinetic analysis of covalent hybrid plasminogen activators: effect of CNBr-degraded fibrinogen on kinetic parameters of Glu1-plasminogen activation

The kinetic parameters of three activator species of Glu1-plasminogen (Glu1-Plg) were compared in their reaction at pH 7.4 and 37 degrees C, in the presence and absence of CNBr-digested fibrinogen (CNBr-Fg). The urokinase- (u-PA-) derived covalent hybrid activator PlnA-u-PAB had an apparent Michaelis constant (Kplg) of 7.44 microM, a catalytic rate constant (kplg) of 51.1 min-1, and a second-order rate constant (kplg/Kplg) of 6.87 microM-1 min-1. The tissue plasminogen activator (t-PA) derived covalent hybrid activator PlnA-t-PAB was characterized by a Kplg of 3.33 microM, a kplg of 1.03 min-1, and a kplg/Kplg of 0.309 microM-1 min-1. The kplg/Kplg values for the parent u-PA and t-PA activators were 6- and 16-fold higher than the respective hybrids, mainly due to an approximately 10-fold increase in the apparent Kplg for the hybrids. In the presence of CNBr-Fg, the increase of the kplg/Kplg values for u-PA and its hybrid was 1.1-fold, but for t-PA and its hybrid, the increases were 7- and 12-fold, respectively. In both the absence and presence of CNBr-Fg, activator t-PAB had an apparent Kplg of 19.1 and 27.6 microM and a kplg of 2.9 and 5.0 min-1, respectively. The increase in the kplg/Kplg value with CNBr-Fg was 1.2-fold. The streptokinase- (SK-) derived activators Glu1-plasmin.SK (Glu1-Pln.SK), Val442-Pln.SK, and Val561-Pln.SK had apparent Kplg values of 0.458, 0.268, and 0.121 microM and kplg values of 20.0, 126.0, and 63.3 min-1, respectively. In the presence of CNBr-Fg, the first two activators showed an approximately 1.4-fold increase and the last showed a 1.4-fold decrease in their kplg/Kplg values. The catalytic efficiency (kplg/Kplg) of the various activator species fell in the decreasing order SK greater than u-PA greater than t-PA, in either the presence or absence of CNBr-Fg. CNBr-Fg enhanced significantly the activities of only two activators, t-PA and PlnA-t-PAB.